Oxygen Consumption of Single Bovine Embryos Probed by Scanning

Jun 27, 2001 - Regional Joint Research Project of Yamagata Prefecture, Japan Science and Technology Corporation,. 2-2-1 Matsuei, Yamagata 990-2473, Ja...
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Anal. Chem. 2001, 73, 3751-3758

Oxygen Consumption of Single Bovine Embryos Probed by Scanning Electrochemical Microscopy Hitoshi Shiku,*,† Takuo Shiraishi,† Hiroaki Ohya,†,‡ Tomokazu Matsue,†,§ Hiroyuki Abe,†,| Hiroyoshi Hoshi,†,| and Masato Kobayashi†,⊥

Regional Joint Research Project of Yamagata Prefecture, Japan Science and Technology Corporation, 2-2-1 Matsuei, Yamagata 990-2473, Japan, Institute for Life Support Technology, 2-2-1 Matsuei, Yamagata 990-2473, Japan, Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aramaki Aoba, Sendai 980-8579, Japan, Research Institute for the Functional Peptides, 4-3-32 Shimojo, Yamagata 990-0823, Japan, and Yamagata Agricultural Research and Training Center, 1076 Torigoe, Shinjo 996-0041, Japan

Oxygen consumption of individual bovine embryos was noninvasively quantified by scanning electrochemical microscopy (SECM). A probe microelectrode was used to scan near a single embryo surface in a culture medium to monitor the oxygen reduction current at 37 °C, under a water-saturated atmosphere of 5% CO2 and 95% air. The oxygen concentration profiles near the embryos were in good agreement with the theoretical spherical diffusion. When an embryo reached the stage of a morula with a 74µm radius on day 6 after in vitro fertilization, the oxygen concentration difference (∆C) between the bulk solution and the morula surface was 6.90 ( 1.35 µM. The oxygen consumption rate (F) of the single morula was estimated to be (1.40 ( 0.27) × 10-14 mol s-1. After the SECM measurement, the embryo was continuously cultured for another 2 days and grew to the stage of a blastocyst with a 100-µm radius. For the blastocyst, the ∆C values for the inner cell mass side and the trophoblast side were 16.40 ( 1.83 and 9.14 ( 1.68 µM, respectively. The oxygen consumption rate of the blastocyst was found to be in the range of (2.50 ( 0.46) × 10-14 mol s-1 < F < (4.49 ( 0.50) × 10-14 mol s-1. We have carried out SECM measurements for 19 embryos, and the results were compared in detail with these from an optical microscopic observation. The ∆C values for the morulae on day 6 after in vitro fertilization were strongly related to the morphological embryo quality. The morulae showing a larger ∆C value developed into blastocysts of a larger size, and the ∆C value after the subsequent 2 days of cultivation was found to be increased. In vitro culture techniques of mammalian oocyte maturation, fertilization, and embryo development have been advanced in recent years. The techniques are useful not only to study the mechanisms of early embryogenesis but also to yield a large number of good-quality embryos for practical uses such as embryo †

Japan Science and Technology Corp. Institute for Life Support Technology. § Tohoku University. | Research Institute for the Functional Peptides. ⊥ Yamagata Agricultural Research and Training Center. ‡

10.1021/ac010339j CCC: $20.00 Published on Web 06/27/2001

© 2001 American Chemical Society

transfer and the production of cloned and transgenic animals. One of the final targets is to produce in vitro-derived and genetically modified embryos that can ensure a high rate of pregnancy. Many technical challenges have been met to improve the embryo quality. In most mammalian species including cows, the embryos at the blastocyst stage are most attractive because they are usually transferred into recipient animals without any surgical operation. Through the development from a one-cell embryo to the blastocyst stage, the metabolic processes drastically change because of genome activation and a large increase in protein synthesis. Radioisotope labeling techniques have been widely used to quantify glucose, pyruvate, lactate, carbon dioxide, or amino acids in metabolic pathways, and detection of metabolites originating from a single embryo has already been attempted.1-5 Oxygen consumption is another ubiquitous parameter to add more valuable information in metabolic mechanisms. Oxygen consumption of mammalian embryos has been studied for more than forty years with various methods such as Cartesian diver,6,7 spectrophotometric,8-10 fluorescence,11-13 and electrochemical techniques.14-19 Currently, it is of great interest to noninvasively (1) Brinster, R. L. Exp. Cell Res. 1967, 47, 271-277. (2) Wales, R. G. J. Reprod. Fertil. 1986, 76, 717-725. (3) Javed, M. H.; Wright, R. W., Jr. Theriogenology 1991, 35, 1029-1037. (4) Rieger, D.; Loskutoff, N. M.; Betteridge, K. J. Reprod. Fertil. Dev. 1992, 4, 547-557. (5) Khurana, N. K.; Nieman, H. Biol. Reprod. 2000, 62, 847-856. (6) Fridhandler, L.; Hafez, E. S.; Pincus, G. Exp. Cell Res. 1957, 13, 132-139. (7) Mills, R. M., Jr.; Brinster, R. L. Exp. Cell Res. 1967, 47, 337-344. (8) Magnusson, C.; Hillensjo, T.; Tsafriri, A.; Hultborn, R.; Ahren, K. Biol. Reprod. 1977, 17, 9-15. (9) Nilsson, B. Magnusson, C. Widehn, S.; Hillensjo, T. J. Embryol. Exp. Morphol. 1982, 71, 75-82. (10) Magnusson, C.; Hillensjo, T.; Hamberger, L.; Nilsson, L. Hum. Reprod. 1986, 1, 183-184. (11) Houghton, F. D.; Thompson, J. G.; Kennedy, C. J.; Leese, H. J. Mol. Reprod. Dev. 1996, 44, 476-485. (12) Thompson, J. G.; Partridge, R. J.; Houghton, F. D.; Cox, C. I.; Leese, H. J. J. Reprod. Fertil. 1996, 106, 299-306. (13) Donnay, I.; Leese, H. J. Mol. Reprod. Dev. 1999, 53, 171-178. (14) Overstrom, E. W.; Duby, R. T.; Dobrinski, J.; Roche, J. F.; Boland, M. P. Theriogenology 1992, 37, 269. (15) Benos, D. J.; Balaban, R. S. Biol. Reprod. 1980, 23, 941-947. (16) Smith, P. J. S.; Hammar, K.; Porterfield, D. M., Sanger, R. H.; Trimarchi, J. R. Microsc. Res. Tech. 1999, 46, 398-417. (17) Land, S. C.; Porterfield, D. M.; Sanger, R. H.; Smith, P. J. S J. Exp. Biol. 1999, 202, 211-218.

Analytical Chemistry, Vol. 73, No. 15, August 1, 2001 3751

determine the oxygen consumption for monitoring the metabolism. Magnusson et al. investigated the oxygen consumption of individual human oocytes and blastocysts using a microspectrophotometric technique based on monitoring oxyhemoglobin absorbance.10 Leese et al. utilized an ultramicrofluorescence technique based on fluorescence quenching of pyrene in paraffin oil facing a small volume of a medium solution containing murine11 or bovine embryos.12,13 Overstrom et al. studied the oxidative metabolism of individual bovine blastocysts by a multichannel embryo microrespiration system based on oxygen electrodes.14 Most recently, a scanning electrode technique, called the selfreferencing microelectrode technique,16,17 has been employed to quantify oxygen consumption of individual mouse embryos of onecell to blastocyst stages.18,19 We report here a study quantifying the oxygen consumption of single bovine embryos by scanning electrochemical microscopy (SECM), a technique in which the tip of a microelectrode is scanned to monitor the local distribution of electroactive species near the sample surface.20-22 Various biological systems including single living cells have been studied by the SECM.23-33 Our study focuses on the noninvasive nature of SECM, to compare the oxygen consumption value of each single embryo with its morphological features and its developmental potential. The oxygen consumption value of the single, identical embryo has been measured at different developmental stages. Although a scanning probe technique was already applied to mouse embryos, we show several novel results that have not been reported yet: (1) Spatial imhomogeneity at the bovine blastocyst surface can be detected with SECM. (2) Oxygen consumption of an individual bovine morula strongly correlates to its morphology. (3) Oxygen consumption activity of an individual embryo in the earlier developmental stage is strongly correlated with the following development of the embryo in terms of the size and oxygen consumption. EXPERIMENTAL SECTION Embryo Culture. The serum-free media IVD101 and IVMD101 used in the present work were donated by the Research Institute (18) Porterfield, D. M.; J. R. Trimarchi, Keefe, D. L.; Smith, P. J. S. Biol. Bull. 1998, 195, 208-209. (19) Trimarchi, J. R.; Liu, L.; Porterfield, D. M.; Smith, P. J. S.; Keefe, D. L. Biol. Reprod. 2000, 62, 1866-1874. (20) Bard, A. J.; Fan, F.-R. F., Kwak. J. Lev. O. Anal. Chem. 1989, 61, 132-138. (21) Engstrom, R. C.; Pharr, C. M. Anal. Chem. 1989, 61, 1099A-1104A. (22) Mirkin, M. V. Anal. Chem. 1996, 68, 177A-182A. (23) Lee, C.; Kwak, J. Bard, A. J. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 17401743. (24) , Tsionsky, M.; Cardon, Z. G.; Bard, A. J.; Jackson, R. B. Plant Physiol. 1997, 113, 895-901. (25) Yasukawa, T.; Kondo, Y.; Uchida, I.; Matsue, T. Chem. Lett. 1998, 767768. (26) Yasukawa, T.; Uchida, I.; Matsue, T. Electrochemistry (Japan) 1998, 66, 660-661. (27) Yasukawa, T.; Uchida, I.; Matsue, T. Biophys. Biochim. Acta 1998, 1369, 152-158. (28) Yasukawa, T.; Uchida, I.; Matsue, T. Biophys. J. 1999, 76, 1129-1135. (29) Yasukawa, T.; Kaya, T.; Matsue, T. Anal. Chem. 1999, 71, 4637-4641 (30) Leszczyszyn, D. J.; Jankowski, J. A.; Viveros, H. O.; Diliberto, E. J. Jr.; Near, J. A.; Whightman, R. M. J. Biol. Chem. 1990, 265, 14736-14737. (31) , Arbault, S.; Pantano, P.; Jankowski, J. A.; Vuillaume, M.; Amatore, C. Anal. Chem. 1995, 97, 3382-3390. (32) Lu, H.; Gratzl, M. Anal. Chem. 1999, 71, 2821-2830. (33) Jung, S.-K.; Gorski, W.; Aspinwall, C. A.; Kauri, L. M.; Kennedy, R. T. Anal. Chem. 1999, 71, 3642-3646.

3752 Analytical Chemistry, Vol. 73, No. 15, August 1, 2001

Table 1. Summary of Morphological Observations and Oxygen Consumption Values of Bovine Embryosa sample no.

rank of morulae

rs/µm

∆C/µM

F × 1014/mol s-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14

A A A A B A B B B A B B C C

75 75 75 74 73 69 75 75 69 72 70 70 75 69

9.25 ( 1.04 8.77 ( 0.49 6.93 ( 1.40 6.90 ( 1.35 6.71 ( 1.35 5.92 ( 0.81 5.77 ( 0.70 5.68 ( 0.97 5.27 ( 0.79 4.58 ( 1.39 4.46 ( 0.95 4.28 ( 0.74 2.99 ( 0.75 2.22 ( 1.12

1.90 ( 0.21 1.80 ( 0.10 1.42 ( 0.29 1.40 ( 0.27 1.34 ( 0.27 1.12 ( 0.15 1.18 ( 0.14 1.17 ( 0.20 0.996 ( 0.15 0.903 ( 0.27 0.855 ( 0.18 0.820 ( 0.14 0.614 ( 0.15 0.419 ( 0.21

70 70 70 75 72

1.74 ( 0.69 1.22 ( 0.46 1.04 ( 0.39 0.95 ( 0.24 0.77 ( 0.47

0.333 ( 0.13 0.234 ( 0.09 0.199 ( 0.08 0.195 ( 0.05 0.152 ( 0.09

15 16 17 18 19

a The viable morulae with different morphology were further categorized to ranks A, B, and C (excellent, good, and fair, respectively).

for the Functional Peptides. The formulations of IVD101 and IVMD101 were shown in the literature.34,35 IVD101 was used for a lower oxygen culture (5% O2) and IVMD101 for a high-oxygen culture (20% O2). The procedures of oocyte collection, in vitro maturation and fertilization of oocytes, and in vitro embryo culture were described elsewhere.34,35 After in vitro fertilization, fertilized oocytes were placed in 250-µL droplets of IVD101 medium under paraffin oil on six-well culture plates (Repro C-1 plate, Research Institute for the Functional Peptides) and incubated for 6 days at 38.5 °C in a water-saturated atmosphere of 5% O2, 5% CO2, and 90% N2. Embryos at the morula stage were selected at day 6 after in vitro fertilization and transferred into IVMD101 medium. The cultured embryos were transported from another laboratory within 20 min at ambient temperature before the SECM measurements. After transportation, the embryo samples were immediately incubated at 37 °C in a water-saturated atmosphere of 5% CO2 and 95% air, and the SECM measurements were then carried out within 12 h. Embryos measured by the SECM were additionally incubated at 37 °C in a water-saturated atmosphere of 5% CO2 and 95% air, for further examination of the following development of embryos determined by the SECM and morphological observations. Morulae cultured in IVD101 medium normally consist of 50100 cells (blastomeres). The morulae can be categorized as ranks A, B, and C (excellent, good, and fair, respectively) based on microscopic observation of the morphology.36 Rank A appears spherical and symmetrical with cells of uniform size, color, and texture. Rank B shows trivial imperfections such as a few extruded (34) Yamashita, S.; Abe, H.; Itoh, T.; Satoh, T.; Hoshi, H. Cytotechnology 1999, 31, 121-129. (35) Abe, H.; Yamashita, S.: Itoh, T.; Satoh, T.; Hoshi, H. Mol. Reprod. Dev. 1999, 53, 325-335. (36) Lindner, G. M.; Wright, R. W., Jr. Theriogenology 1983, 20, 407-416.

Figure 1. Local concentration profiles for a morula on day 6 after in vitro fertilization, versus the distance (r) from the embryo surface (A) and the rs/(r + rs) value (B). The tip approached the morula by scanning in the X-, Y-, and Z-directions, as shown in the left, middle, and right portions of the figure. For the X- and Y-directions, the tip scanned a 500-µm step at 14.7 µm/s. For the Z-direction, the tip scanned a 160-µm step at 4.9 µm/s. The measurements were carried out in the culture medium at 37 °C in a water-saturated atmosphere of 5% CO2 and 95% air. The photograph shows the measured morula. The tip microelectrode and the holding pipet are seen on the upper side and the right side of the morula, respectively. Bar, 200 µm. The scheme shows the situations (tip radius, 1.8 µm; seal radius, 4.0 µm; tip-sample distance, 5.6 µm; sample radius, 76.6 µm) approaching the sample surface according to the X-, Y-, and Z-directions.

blastomeres. Rank C is characterized by the presence of many extruded blastomeres. Blastocysts cultured in IVD101 medium showed a clear differentiation between the inner cell mass (ICM) cells and trophoblast (TRP) cells that surrounded the inner cavity of embryos (blastocoel). Blastocysts consist of 100-200 cells and are surrounded by the zona pellucida with thickness of 3-20 µm. The retarded embryos cited in Table 1 are embryos that did not develop to the morula stage on day 6 after in vitro fertilization. SECM Measurement. A single bovine embryo was transferred into a 35-mm-diameter culture dish (Nunc) filled with 3 mL of IVMD 101 medium and placed on an inverted optical microscope stage (Nikon, TE300). The embryo was manipulated and held at the center of the optical microscopic view with a manual micropositioner (You, Ltd., UM-3C) and a manual microinjector (Narishige, IM-26-2). A motor-driven XYZ stage (Chuo Seiki, M9103) was located on the microscope stage for electrode tip scanning. The medium temperature was maintained at 37 °C on the microscope stage equipped with a thermostat flowing water jacket. The measurement instruments were covered with a plastic

sheet (volume, 15 L) and water-saturated 5% CO2 and 95% air gas was allowed to flow. A Pt microdisk electrode sealed with a tapered soft-glass capillary (World Precision Instruments, PG10165-4) was fabricated according to the literature. 37 The tip radii determined with cyclic voltammetry in a 5.0 mM K4Fe(CN)6, 0.1 M KCl solution were 1.8 µm for the experiment in Figure 1 and 0.92 µm for the others. The radius of the tip including the glass seal part was 4.0 µm. For detection of the oxygen reduction current, the tip potential was held at -0.6 V vs Ag/AgCl. Voltammetry of the Pt microdisk in IVMD 101 medium showed a steady-state oxygen reduction wave. No response from other electrochemically active species was observed in the potential range between -0.6 to +0.5 V. We also measured cyclic voltammograms near the surface of a morula, the ICM side, and the TRP side of a blastocyst. Again, no electrochemical species except for oxygen was observed in the potential range between -0.6 to +0.5 V, confirming that oxygen (37) Matsue, T.; Koike, S.; Uchida, I. Biochem. Biophys. Res. Commun. 1993, 197, 1283-1287.

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Figure 2. Plots of the oxygen concentration profile versus the tip-sample distance (r) (A) and the rs/(r + rs) value (B) at 37 °C in a watersaturated atmosphere of 5% CO2 and 95% air. The photographs and the profiles correlate to a morula on day 6 after fertilization (left) and a blastocyst on day 8 (middle and right). The blastocyst was developed from the morula shown on the left, after cultivation for another 2 days. The data were accumulated n ) 8, 8, and 6, for the profiles of the morula (left), the ICM (middle), and the TRP sides (right) of the blastocyst, respectively. Bar, 200 µm. Tip radius, 0.92 µm

is the main species contributing to the reduction current which has been recorded at -0.6 V. To monitor the oxygen reduction current near the single embryo, the tip was scanned back and forth between a point close to the zona pellucida surface and a point 500 µm apart from the embryo surface, along with a horizontal line crossing the center of the embryo. The tip scanned at 14.7 µm/s except for specifically mentioned. The microelectrode disk plane was guided perpendicular to the embryo surface to prevent the tip current from perturbations due to topographic artifacts. The situation tip located close to the embryo can be seen in the photographs and scheme in Figures 1 and 2. When the tip located close to the sample surface, the distance between the tip and the embryo surface was typically ∼5 µm. The exact distance was determined from the photograph taken for each measurement. During the measurements, microscopic observation was essential to confirm that the positions of the tip and the sample were appropriate. Estimation of Oxygen Consumption Based on Spherical Diffusion. When a sample with a spherical shape is placed in a solution and consumes oxygen at a steady-state rate, the oxygen 3754

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concentration distribution according to the distance (r) from the sample surface is expressed as follows, under the assumption that the oxygen concentration at the surface of the spherical sample is uniform,

C(r) ) (Cs - C*)rs/(r + rs) + C*

(1)

where rs is the radius of the sample and C* and Cs are the concentrations of oxygen in the bulk solution and at the sample surface, respectively. C* is 0.209 mM in aqueous solution at 37 °C in 5% CO2 and 95% air. Cs is determined from the intercepts at rs/(r + rs) ) 1 of C(r) vs rs/(r + rs) plots. The oxygen consumption rate (F) can be expressed by

F ) 4πrsD(C* - Cs)

(2)

where D is the diffusion coefficient of oxygen, 2.18 × 10-5 cm2 s-1 at 37 °C in aqueous solution. In this study, the oxygen concentration difference between the bulk and sample surface (∆C ) C* - Cs) and the oxygen

consumption rate of a single sample (F) were estimated according to the spherical diffusion theories. We repeatedly scanned the electrode back and forth more than three times to estimate the average ( standard deviation (n g 6) of the ∆C for each sample. Although the experimental conditions are not exactly ideal, the equations shown above are useful for quantitatively estimating the mass-transfer rates for oxygen. Studies adopting the spherical diffusion theory to estimate the mass-transfer rate for redox species in various experimental systems have been reported.26,28,38-40 Before measuring oxygen consumption of bovine embryos, we checked several experimental conditions for adopting the spherical diffusion theory to our experimental system. For primary tests of oxygen consumption measurement, protoplasts of green algae (Bryopsis plumosa) were prepared according to previous papers26-28 and measured in the dark. The ∆C estimated by a scanning tip at 14.7 µm/s was identical within 10% deviation to that obtained by a holding tip at 12 points in a 500-µm scan rage. The linearity of the C(r) vs rs/(r + rs) plots for the protoplasts with radii of 56.3 and 159.9 µm was very good. The correlation coefficients (R) were 0.987 and 0.999, respectively. The disturbances of the concentration profiles by tip scanning are negligible even for the samples with radius larger than 100 µm. RESULTS AND DISCUSSION In the present study, the oxygen consumption rates of a single bovine embryo are quantified based on the spherical diffusion theory. The sample radius (rs) is defined as the embryo radius including the zona pellucida. Even though the shape of the embryo mass in the morulae is not ideally spherical sometimes, the shape including the zona pellucida is sufficiently sphere-shaped. The oxygen distribution within the embryo or the oxygen permeation process through the zona pellucida is very interesting; however, we do not focus on the oxygen concentration of the inner part of the sample at the present research stage. We experimentally examined the oxygen concentration profiles near a bovine embryo according to the different directions, to investigate the homogeneity of the oxygen concentration at the embryo surface, the interface between the sample and the medium solution. A partial blocking effect of oxygen diffusion due to the tip microelectrode being very close to the sample surface is also discussed. Figure 1 shows oxygen concentration profiles for a morula on day 6 after in vitro fertilization versus the tip-sample distance (r) (Figure 1A) and the rs/(r + rs) value (Figure 1B). The tip approached the morula according to the X-, Y-, and Z-directions, as shown in the left, middle, and right of the figure, respectively. The radius of the morula including the zona pellucida (rs) was 76.6 µm. The measurements were carried out in the culture medium at 37 °C in a water-saturated atmosphere of 5% CO2 and 95% air. As shown in Figure 1A, the oxygen concentration decreased near the morula surface. The decrease originates from oxygen consumption, mainly respiration, by the single morula. Only in the profile in the Z-direction was a steep decline observed when the tip-sample distance was less than ∼10 µm. The decrease results from partial blocking of the oxygen diffusion to the tip electrode surface (called negative-feedback effect), which (38) Bath, B. D.; Lee, R. D.; White, H. S. Anal. Chem. 1998, 70, 1047-1058. (39) Scott, E. R.; White, H. S.; Phipps, J. B. Anal. Chem. 1993, 65, 1537-1545. (40) Horrocks, B. R.; Mirkin, M. V.; Pierce, D. T.; Bard, A. J.; Nagy, G.; Toth, K. Anal. Chem. 1992, 64, 1795-1803.

was not observed in the case where the tip was scanned in the Xand Y-directions. As schematically shown in Figure 1, the seal part of the tip physically prevents the tip from locating the region where the negative-feedback effect is expected. In the plots of the oxygen concentration versus the rs/(r + rs) value (Figure 1B), the lines are given by the method of least squares. For the profile in the Z-direction only, data for a distance less than 18 µm were neglected in estimating the oxygen concentration difference between the bulk and the sample surface (∆C). The three ∆C values were coincident: 4.36 ( 0.46, 4.14 ( 0.38, and 4.50 ( 0.51 µM for the X-, Y-, and Z-directions, respectively. The correlation coefficients (R) for the X-, Y-, and Z-directions were 0.983, 0.959, and 0.978, respectively. We further measured the concentration distribution at the morula surface for 10 samples. The ∆C values for the tip scanned in the different directions of a morula were identical with 0-15% variations. The results partly support the homogeneity of the oxygen concentration at morula surfaces. The zona pellucida (with ∼20-µm thickness for a morula) presumably weaken the spatial concentration differences at the outer surface of the embryo. This scanning electrochemical measurement procedure enables quantifying the oxygen consumption of single embryos without any damage to the sample. Because there is no need to add labeling materials, the measurement conditions can be ideally close to the embryo culture conditions. Figure 2 shows oxygen concentration profiles versus the tip-sample distance (r) (Figure 2A) and the rs/(r + rs) value (Figure 2B), measured for the same embryo at different developmental stages. First, a profile was measured for a morula with a 74-µm radius (left in Figure 2) on day 6 after in vitro fertilization. The morula was incubated for a further 2 days at 37 °C in a water-saturated atmosphere of 5% CO2 and 95% air and developed to the blastocyst stage with a 100-µm radius (middle and right in Figure 2). The measurements were then carried out for the ICM side (middle in Figure 2) and the TRP side (right in Figure 2) of the blastocyst. The oxygen concentration decrease for the ICM side of the blastocyst was significantly larger than that for the morula. For the TRP side, the decrease was not as large as that for the ICM side. Both profiles for the blastoctyst were more widely spread to the bulk solution, compared with that for the morula, clearly indicating that the profile depends on the embryo radius. Figure 2B shows oxygen concentration profiles as a function of the rs/(r + rs) value. Each profile exhibits excellent linearity, leading us to suppose that the concentration distribution near the embryo is well defined by the spherical diffusion theory as shown in the eq 1. Each line in Figure 2B is given by the method of least squares. The correlation coefficients (R) for the morula, the ICM side, and the TRP side of the blastocyst were 0.942, 0.995, and 0.977, respectively. As the concentration profile near the surface distributes in much wider length (∼500 µm) compared with the dimension of the tip radius (0.92 µm), current perturbation due to oxygen diffusion blocking is ruled out. We repeatedly scanned the electrode back and forth more than three times to estimate the average ( standard deviation (n g 6) for each sample. The ∆C value for the morula with a 74-µm radius was 6.90 ( 1.35 µM. The total oxygen consumption rate of the single embryo (F) can be estimated by eq 2 and was found to be (1.40 ( 0.27) × 10-14 mol s-1. For the blastocyst, ∆C for the ICM Analytical Chemistry, Vol. 73, No. 15, August 1, 2001

3755

Figure 3. Photographs of the morulae on day 6 after in vitro fertilization, cited as 1-14 in Table 1. Bar, 200 µm.

side (∆CICM) was 16.40 ( 1.83 µM and that for the TRP side (∆CTRP) was 9.14 ( 1.68 µM. Even though the plots for the blastocyst in Figure 2B show very good linearity, the boundary conditions on the blastocyst might not be appropriate for adopting eq 2 because the surface concentration of the blastocyst is considered to be inhomogeneous. Therefore, the fluxes obtained by substituting ∆CICM and ∆CTRP into eq 2 indicate the upper and lower limits of the oxygen consumption rate per single blastocyst, and the F value is in the range of (2.50 ( 0.46) × 10-14 mol s-1 < F < (4.49 ( 0.50) × 10-14 mol s-1. The inhomogeneity at the surface of a blastocyst were further investigated. For 9 out of 13 blastocysts, the differences between the ∆CICM and the ∆CTRP were detected ((∆CICM/∆CTRP) ) 1.22-1.96). For the other four, the inhomogeneity at the blastocyst surface was not detected ((∆CICM/∆CTRP) ) 1.00-1.06). The average radius of the former nine blastocysts was 92 µm and that for the latter four was 81 µm. The ∆C difference at a blastocyst surface tends to be larger for blastocysts of relatively larger sizes. We applied the measurement and the analysis procedures mentioned above to characterize many embryos. The results are summarized in Table 1 for 19 embryos (morulae and retarded embryos) on day 6 after in vitro fertilization. Embryos are listed in order of larger ∆C. The embryo radii including zona pellucida 3756

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(rs), and the oxygen consumption rates per single embryos (F) calculated by eq 2 are also cited. Sample 4 is the embryo shown in Figure 2. Figure 3 shows the optical microscopic images of the morulae cited in Table 1. For the morulae, the ∆C value is a useful parameter for indicating the oxygen consumption of single embryos compared with the F value, because the radii (rs) of the morulae are very similar (69-77 µm). For the morulae (1-14), the ∆C values were distributed over a wide range from 2.22 to 9.25 µM. On the basis of microscopic observation of the morphology,36 the morulae samples can be categorized as ranks A, B, and C (excellent, good, and fair, respectively). The ∆C values for the morulae in rank A varied but were at a higher level (4.58-9.25 µM) than that for the other morulae (Table 1). The ∆C value apparently tends to be smaller as the rank of morula is lower. The retarded embryos (15-19) showed considerably smaller ∆C values compared with the morulae in ranks A, B, and C. These results indicate that the oxygen consumption of a single morula strongly correlates with its morphological observation. The system used in the present study detects as small as a 1 µM difference in oxygen concentration; therefore, the ∆C value gives a precise and quantitative feature of a single embryo. In the following section, we show that the ∆C value is a significant indicator of viability and further developmental potential of an individual embryo.

Table 2. Relationship between the Oxygen Consumptions of the Morulae on Day 6 and the Further Developmental Potency of These Embryos (n ) 29)a category determined by SECM measurement rank A (n ) 13) ∆C(day 6) g 5.0 µM ∆C(day 6) < 5.0 µM rank B (n ) 13) ∆C(day 6) g 5.0 µM ∆C(day 6) < 5.0 µM rank C (n ) 3) ∆C(day 6) g 5.0 µM ∆C(day 6) < 5.0 µM total (n ) 29) ∆C(day 6) g 5.0 µM ∆C(day 6) < 5.0 µM

rs < 80

rs (day 8) 80 e rs < 90

90 e rs

blastocoel formation

development to hatched blastocyst

2/12 0/1

1/12 1/1

9/12 0/1

12/12 1/1

9/11 0/1

2/8 5/5

4/8 0/5

2/8 0/5

7/8 1/5

4/8 0/5

0/0 2/3

0/0 1/3

0/0 0/3

0/0 1/3

0/0 0/3

4/20 (20%) 7/9 (78%)

5/20 (25%) 2/9 (22%)

11/20 (55%) 0/9 (0%)

19/20 (95%) 3/9 (33%)

13/19 (68%) 0/9 (0%)

a The embryo radius including zona pellucida (r ) on day 8, the blastocoel formation, and the development to hatched blastosyst was surveyed s under an optical microscope. Judgment based on the oxygen consumption is also adopted for the ranks A, B, and C.

Figure 4. Relation between the ∆C on day 6 after in vitro fertilization and the rs on day 8 for individual embryos. The line is given by the method of least squares. The bar indicates the standard deviations.

Figure 4 shows the relation between the ∆C on day 6 and the rs on day 8 after in vitro fertilization for individual embryos, representing a reasonable correlation between oxygen consumption of the morula and its developmental potential. The line in Figure 4 was obtained by the method of least squares and the correlation coefficient (R) was 0.619. Even if the oxygen consumption simply reflects the cell number or the amount of mitochondria in a single embryo, it is an important parameter to quantify the viability and the developmental capacity of individual embryos. It should be emphasized here that the oxygen consumption can be precisely determined using the method described in the present paper. Table 2 summarizes the results (n ) 29) of ∆C on day 6 morulae measured by the SECM and the following developmental parameters obtained by morphological observations in the further incubation, the rs on day 8, the blastocoel formation, and the development to hatched. For morulae with a ∆C of less than 5.0 µM on day 6, they grew only slightly. On day 8, the radius of 78% of the embryos was smaller than 80 µm. The blastocoel formation was only 33%, and no hatched blastocyst was observed. On the contrary, for the morulae with a ∆C of more than 5.0 µM on day 6, 80% of the embryos became bigger with a radius larger than

Figure 5. Relation between F on day 6 after in vitro fertilization and F on day 8 for individual embryos. The line is given by the method of least squares. The F values were calculated by eq 2. Concerning F on day 8, the filled squares, the filled circles, and the open circles were given by substituting ∆C of the morulae, ∆CICM of the blastocysts, and ∆CTRP of the blastocysts, respectively. The bar indicates the standard deviations.

80 µm on day 8. Furthermore, the blastocoel and hatched blastocyst formation reached 95 and 68%, respectively. The judgment is valid even in the same morphological categories, especially in rank B. The ratio of the blastocoel and hatched blastocyst formation on the morulae in rank B with a ∆C of more than 5.0 µM is much higher than those with a ∆C of less than 5.0 µM. Figure 5 shows the relation between the oxygen consumption rates (F) on days 6 and day 8 for single embryos. The oxygen consumption rates per a single embryo (F) were calculated based on eq 2. The filled squares indicate the F values of embryos remaining at the morula stage on day 8. Filled and open circles mean the F values determined using ∆CICM and ∆CTRP of blastocysts, respectively. Again, oxygen consumption rates for individual embryos on days 6 and 8 were found to be strongly correlated. The line in the figure was given by the method of least squares and the correlation coefficient (R) was 0.635. The F value on day 8 increases by 1.5-2.0-fold compared with that on day 6. The F values for morulae on day 6 and blastocysts on day 8 are found to be (0.614-1.90) × 10-14 and (0.837-4.49) × 10-14 Analytical Chemistry, Vol. 73, No. 15, August 1, 2001

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mol s-1, respectively. The oxygen consumption rates of individual bovine embryos have been estimated by several groups. Leese et al.12,13 reported that the oxygen consumption values for a compact morula on day 4, blastocyst on day 5, and expanded blastocyst on day 8 were 0.48 × 10-14, 1.12 × 10-14, and 2.53 × 10-14 mol s-1, respectively. Their values are comparable to our results, considering the difference in the measurement principles and the experimental conditions. Finally, we will discuss the applicability of the oxygen consumption of single embryos for judging the viability and the developmental potency of the embryos. Some predictive values of metabolic parameters for embryo viability have been reported to be superior to morphological criteria in embryo selection.41-43 Leese et al. reported a fluorescence technique to monitor the oxygen consumption,11-13 as well as glucose uptake or other molecular species produced from mammalian embryos.42,43 They measured the oxygen consumption of more than two embryos together and required 4-6 h for the measurement.11,12 To discriminate a qualified embryo, the detection system should be applicable to a single embryo and be rapid in measurement. Magnusson et al. succeeded in determining the oxygen consumption of individual human blastocysts with a microspectrophotometric technique.10 They observed a correlation between the oxygen consumption and the morphological quality. However, they did not clarify the correlation of oxygen consumption with the survival rate of the embryos. Overstrom et al. presented a criterion of judgment in embryotransfer experiments utilizing oxygen electrodes.14 According to their results, bovine blastocysts with oxygen consumption larger than 2.48 × 10-14 mol s-1 showed a 9% higher survival rate than those with smaller oxygen consumption, during the process before and after freezing. However, the difference in oxygen consumption rates between morulae and blastocysts was not revealed. The methods mentioned above rely on the principle of monitoring the concentration changes of the bulk solution, which is inappropriate for rapid quantitative measurements. The correlation among the oxygen consumption values, morphological features, and developmental potential is not very clear. The SECM procedure, instead, can directly measure the concentration profiles within a few minutes to estimate the oxygen consumption rates by utilizing (41) Rondeau, M.; Guay, P.; Goff, A. K.; Cooke, G. M. Theriogenology 1995, 44, 351-366. (42) Leese, H. J.; Barton, A. M. J. Reprod. Fertil. 1984, 72, 9-13. (43) Gardner, D. K.; Leese, H. J. J. Exp. Zool. 1987, 242, 103-150. (44) Kaya, T.; Nishizawa, M.; Matsue, T., unpublished results.

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the spherical diffusion theory. The SECM procedure also possesses the spatial resolution that has made it possible for the first time to sense the inhomogeneous oxygen concentration at the blastocyst surface. SECM also allows the measurements without any additions of labeling reagent. Very recently, a scanning electrochemical technique was applied to mouse embryos in one-cell to blastocyst stages by Trimarchi et al.19 In their method, the tip was oscillated at 0.3 Hz with 10-µm amplitude to lower the detection limit to 0.01 µM. They noted the noninvasiveness of the technique and showed that more than 90% of two-cell embryos normally developed to blastocysts. However, they did not show the correlation between the oxygen consumption and the developmental potential. For detailed quantification of embryos, it is necessary to measure the ICM side and the TRP sides of blastocysts. In addition, the zona pellucida of the embryo was enzymatically removed in their most experiments, and therefore, the results may not reflect the accurate oxygen consumption of the embryo. In the present study, we have applied SECM to measure oxygen consumption of single bovine embryos. The oxygen consumption has been monitored at different developmental stages of a single, identical bovine embryo. For the morulae, the relation between oxygen consumption and morphological quality is discussed in detail. The morulae with the larger oxygen consumption on day 6 developed into blastocysts of larger sizes and larger oxygen consumption on day 8 after continuous cultivation. The morula with larger oxygen consumption has strong potentiality for further development into blastocoel and hatched blastocyst formation. Even though many microamperometric studies have already been reported for probing various cell functions at singlecell levels, our study showed the high potential of the SECM procedure to noninvasively assess the viability of living samples. As SECM is also applicable for multisample analysis in a small area in the imaging mode,25,44 a high-throughput and semiautomatic measuring system will be achieved in the future. ACKNOWLEDGMENT This work was partly supported by Grants-in-Aid for Scientific Research (11450323, 11227201) from the Ministry of Education, Science and Culture, Japan.

Received for review March 21, 2001. Accepted May 1, 2001. AC010339J